We have achieved significant enhancement of gene delivery into livers of large animals using ultrasound (US)-targeted microbubble (MB) destruction methods. An infusion of pGL4 (encoding a luciferase reporter gene) plasmid DNA (pDNA) and MBs into a portal-vein segmental branch of a porcine liver was exposed to US for 4 min. Therapeutic US induced cavitation of MBs to temporarily permeabilize the vascular endothelium and cell membranes, allowing entry of pDNA. We obtained a 64-fold enhancement in luciferase expression in pig livers compared to control without US using an unfocused, dual-element transducer (H105, center frequency [f(c)] = 1.10 MHz) at 2.7 MPa peak negative pressure (PNP). However, input electrical energy was limited, and modified transducers were designed to have spherical (H185A, f(c) = 1.10 MHz) or cylindrical foci (H185B, f(c) = 1.10 MHz; H185D, f(c) = 1.05 MHz) to enhance PNP output. The revised transducers required less electrical input to achieve 2.7 MPa PNP compared to H105, thereby allowing PNP outputs of up to 6.2 MPa without surpassing the piezo-material limitations. Subsequently, luciferase expression significantly improved up to 9,000-fold compared to controls with minor liver damage. These advancements will allow us to modify our current protocols toward minimally invasive US gene therapy.

Previously we demonstrated that ultrasound (US)-mediated gene delivery (UMGD) can significantly enhance reporter gene transfer into the mouse and rat livers. This nonviral gene transfer strategy can bypass many obstacles encountered by viral gene therapy. Most significantly, we have achieved therapeutic levels of FVIII following UMGD into hemophilia A mice. Recently we have successfully developed prototype US systems including several unfocused and semi-focused transducers to treat large tissue volumes in canine and swine. In order to facilitate the translation of this technology to treat hemophilia, we have recently treated 2 normal dogs with UMGD of FVIII plasmids using an open surgery procedure. Four mg of a high-expressing, liver-specific pBS-HCRHPI-FVIIIA plasmid and 3 ml of Definity® MBs in 8 ml total PBS solution were injected via the segmental portal vein branch with simultaneous exposure of the target liver lobe to therapeutic US (1.1 MHz frequency, 20 cycle pulses, 50 Hz pulse repetition frequency) for 4 minutes using the large diameter transducers. A sham-treated dog received an equivalent pGL4/MB dose, but was not exposed to tUS. We used an apodized dual element unfocused transducer H105 at 2.5Mpa peak negative pressure (PNP) in the first dog experiment and observed low levels of hFVIII gene expression in the treated liver lobes. The second dog experiment was performed using a cylindrically 6.2 MPa at the focal area. One day following treatment, the treated liver lobes were sectioned, and representative sections were fixed and stained for FVIII expression by histochemical staining using a polyclonal anti-FVIII antibody. Untreated normal dog liver and human liver were used as negative and positive controls. Significant hFVIII gene expression was obtained in both treated liver lobes with the expression levels slightly lower than control human liver lobe. Furthermore, fairly homogeneous distribution of FVIII gene expression was observed in hepatocytes. Next, we performed Western blot analysis to confirm if the staining is specific to human FVIII protein. A heavy chain band specific to hFVIII was observed in treated dog plasma and positive hFVIII control, but not in control normal dog plasma and pre-bleed plasma sample from the dog before treatment. Significant enhancement of FVIII-specific clotting activity was also obtained in the second treated dog compared to pre-treatment and the first treated dog. In addition, transaminase levels and histology analysis indicated minimal tissue damage in treated dog livers. Enhancement of FVIII gene expression by UMGD is currently evaluated in hemophilia dogs. Our results sugges that UMGD has great potential for therapeutic treatment of hemophilia A.

This project implements previously developed techniques for guiding and monitoring thermal HIFU therapy using imaging arrays with a spherically shaped therapeutic array for dual-mode operation. Three-dimensional motion tracking, thermal strain measurement, and radiation force rebound measurement within the HIFU steering volume are performed using only the therapy array. The techniques have typically been studied for conventional ultrasound imaging using linear, curvilinear, or phased arrays which are usually placed coaxially in the center of the HIFU array. While the dual-mode approach does not provide the image quality and wide field of view of an imaging array, the perfect co-registration of the therapy array, and its 3D view of the region of interest present a valuable opportunity for ultrasound-guided focused ultrasound (USgFUS) therapy systems. A family of Sonic Concepts broadband spherically-focused HIFU array transducers, driven with the Verasonics HIFU-configured ultrasound system, is used to assess real-time performance of the dual-mode approach, in simulation and experiment. Simulations using the Verasonics acquisition software with a set of point scatterers produce the RF backscatter data used by the monitoring algorithms. The particles are displaced in time using motions that mimic the true or apparent displacements of tissue-like media either in translation, heated by a HIFU focus, or deformed by a radiation force beam. Preliminary experiments using a 128-element HIFU array and a scattering hydrogel phantom indicate that the simulations are effective for studying a range of tradeoffs in transducer design and therapy monitoring approaches.

High-intensity focused ultrasound (HIFU) is a promising technology for non-invasive and minimally invasive ablation of benign and malignant tumors. In HIFU therapy, ultrasound energy is focused within the body to induce thermal denaturation of tissue at the focus without affecting surrounding organs. Recent studies also found that the mechanical effect of HIFU may help to enhance drug delivery and stimulate an anti-tumor immune response in certain tumors, including pancreatic tumors. However, targeting of pancreatic tumors using an extracorporeal source is often not possible due to lack of an adequate acoustic window through overlying bowel gas. The major challenges that complicate HIFU ablation of pancreatic tumors are the presence bowel gas, respiratory motion, and the absence of monitoring. Bowel gas can obstruct the acoustic window for HIFU energy delivery, leading not only to incomplete ablation of the target, but also to thermal bowel injury due to rapid heat deposition at the gas-tissue interface. Respiratory motion during HIFU therapy leads to redistribution of acoustic energy over the target area larger than intended, with possible resultant damage to adjacent tissues. The development of an endoscopic ultrasound (EUS)-guided HIFU transducer enables avoidance of both bowel gas blockage and respiration motion redistribution and provides a reliable monitoring method. EUS-guided HIFU also has many potential benefits including improved targeting, decreased energy requirements and decreased potential for injury to intervening structures.

This paper describes a series of high intensity focused ultrasound (HIFU) phased array transducers with a single Archimedean spiral close packed element arrangement on a spherical surface extending from the central axis outward. Simulation and measurement reveal using the spiral element arrangement on a spherical bowl reduces grating side lobes while maximizing power density at the focus.

Ultrasound (US) was applied to a targeted canine liver lobe simultaneously with injection of plasmid DNA (pDNA)/microbubble (MB) complexes into a portal vein (PV) segmental branch and occlusion of the inferior vena cava (IVC) to facilitate DNA uptake. By using a 1.1 MHz, 13 mm diameter transducer, a fivefold increase in luciferase activity was obtained at 3.3 MPa peak negative pressure (PNP) in the treated lobe. For more effective treatment of large tissue volumes in canines, a planar unfocused transducer with a large effective beam diameter (52 mm) was specifically constructed. Its apodized dual element configuration greatly reduced the near-field transaxial pressure variations, resulting in a remarkably uniform field of US exposure for the treated tissues. Together with a 15 kW capacity US amplifier, a 692-fold increase of gene expression was achieved at 2.7 MPa. Transaminase and histology analysis indicated minimal tissue damage. These experiments represent an important developmental step toward US-mediated gene delivery in large animals and clinics.

Ultrasound (US)-targeted microbubble (MB) destruction (UTMD) can significantly enhance gene delivery in mouse livers when pDNA/MBs were injected into the portal vein (PV) with simultaneous US exposure using a focused transducer. However, this transducer was ineffective in enhancing gene transfer into rats. A 13-mm diameter unfocused transducer was designed and the delivery route of pDNA/MBs was modified into a specific liver lobe, resulting in >100-fold increase in luciferase expression in rats. To facilitate the translation into human application, many technical issues were explored in large animal models. We applied 1.1 MHz US to a targeted canine liver lobe with simultaneous injection of pDNA/MBs into a PV segmental branch and occlusion of the inferior vena cava. For more effective treatment of large tissue volumes, a 52-mm planar unfocused transducer was specifically constructed. Its apodized dual element configuration greatly reduced the near field transaxial pressure variations, producing a uniform field of US exposure for the treated tissues. Together with a 15 kW-capacity US amplifier, a 692-fold increase of gene expression in canines was achieved at 2.7-MPa. Transaminase levels and histology analysis indicated minimal tissue damage. These results demonstrated that UTMD is highly promising for safe and efficient gene delivery into the liver.

This paper describes a novel therapeutic high intensity non-focused ultrasound (HIU) transducer designed with uniform pressure distribution to aid in accelerated gene transfer in large animal liver tissues in vivo. The underlying HIU transducer was used to initiate homogeneous cavitation throughout the tissue while delivering up to 2.7 MPa at 1.1 MHz across its radiating surface. The HIU transducer was built into a 6 cm diameter x 1.3 cm tall housing ergonomically designed to avoid collateral damage to the surrounding anatomy during dynamic motion. The ultrasound (US) radiation was applied in a 'paintbrush-like' manner to the surface of the liver. The layers and geometry of the transducer were carefully selected to maximize the active diameter (5.74 cm), maximize the electrical to acoustic conversion efficiency (85%) to achieve 2.7 MPa of peak negative pressure, maximize the frequency operating band at the fundamental resonance to within a power transfer delta of 1 dB, and reduce the pressure delta to within 2 dB across the radiating surface. For maximum peak voltage into the transducer, a high performance piezoceramic was chosen and a DC bias circuit was built integral to the system. An apodized two element annular pattern was made from a single piezoceramic element, resulting in significant pressure uniformity enhancement. In addition to using apodization for pressure uniformity, a proprietary multi-layered structure was used to improve efficiency while sustaining an operating band from 900 kHz to 1.3 MHz. The resultant operating band allowed for dithering techniques using frequency modulation. The underlying HIU transducer for use in large animals enhances gene expression up to 6300-fold.

In order to fully characterize transducers used in high-intensity focused ultrasound, it is useful to measure the source field at the transducer’s radiating surface while the transducer is radiating into water. A new, ruggedized hydrophone has been recently developed to enable direct-contact scanning at the radiating surface without damage to the source transducer. The hydrophone enables the acquisition of high-resolution images of the source fields for single-element and array transducers. This allows beam features such as unexpected side lobes or other anomalies to be traced back to local conditions at the radiating surface. The compact size of the hydrophone also allows source field measurements to be made in the presence of reflective structures located within the field of the transducer. This hydrophone is useful in the testing of transducer quality. In array transducers, it is also useful in verifying the geometric locations, radiating areas, and channel numbering of array elements, and in the measurement of channel-to-channel crosstalk.